Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.     

Studies of individual carbon sites of hemoglobins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Proton-decoupled natural abundance 13C NMR spectra of carbon monoxide hemoglobins were recorded at 15.18 MHz by the Fourier transform method, under conditions of spectrometer sensitivity sufficient for detection of individual carbon resonances. The aromatic region of each spectrum contains broad bands of methine carbon resonances, and some relatively narrow peaks arising from nonprotonated carbons. Resonances of heme carbons were detected in spectra of carbon monoxide hemoglobins, but not in spectra of ferrihemoglobin (as a result of paramagnetic effects). Spectra of carbon monoxide hemoglobins from various species yielded only a few well resolved individual carbon resonances, most notably those of Cgamma of tryptophan residues. A comparison of the spectra of human adult, human fetal, chicken AII, and bovine fetal hemoglobins yielded specific assignments for all resonances of Cgamma of tryptophan residues. In the cases of human fetal, chicken AII, and bovine fetal hemoglobins, each tryptophan yielded a completely resolved individual carbon resonance. The chemical shift difference between the resonances of Cgamma of Trp-130beta and Cgamma of Trp-37beta is about 6 ppm. The chemical shift difference between Trp A12[14]alpha and Trp A12[15]beta is 1 ppm or less. A comparison of the chemical shifts of analogous tryptophan residues of the four carbon monoxide hemoglobins suggests very similar conformations in solution.     

Studies of individual carbon sites of hen egg white lysozyme by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Assignment of the nonprotonated aromatic carbon resonances to specific residues in the sequence.

The resonances of nonprotonated aromatic carbons in natural abundance 13C NMR spectra of hen egg white lysozyme are assigned to specific residues of the amino acid sequence. Chemical shift considerations, the effect of pH, and partially relaxed Fourier transform NMR spectra are used to assign each resonance to one of the seven types of nonprotonated aromatic carbons of amino acid residues. Spectra of chemically modified lysozyme samples yield various assignments to specific residues in the sequence. Line-broadening effects caused by binding of the relaxation probes Gd3+ and 4-N-acetamido-2,2,6,6-tetramethylipiperidine-1-oxyl yield specific assignments which are fully consistent with those based on chemical modifications. The effects of paramagnetic shift reagents and amino sugar inhibitors do not yield any obvious specific assignments. The effect of pH on the chemical shift of Cgamma of His-15 yields a pKalpha in agreement with published values, and indicates that the imidazole form of His-15 exists mainly (or entirely) as the Nepsilon3-H tautomer. The effect of pH on the chemical shifts (measured up to pH 8.8, at 38 degrees) of Czeta and Cgamma of the 3 tyrosine residues yields crude pKalpha values of 9.5 and 10 for Tyr-23 and one of the other tyrosines, respectively. The 3rd tyrosine residue does not exhibit titration behavior.     

Participation of tryptophan 62 in the self-association of hen egg white lysozyme. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy.

Self-association of hen egg white lysozyme in solution of 38 degrees) is examined by means of natural abundance 13C nuclear magnetic resonance spectroscopy. The effect of pH on the resonances of the nonprotonated aromatic carbons of 9 mM lysozyme, and the effect of protein concentration (at pH 7) on these resonances, both indicate that self-association significantly affects the chemical shift of Cgamma of Trp-62, but not the chemical shifts of the other nonprotonated aromatic carbons. This result is consistent with the reported participation of Trp-62 in the intermolecular contact (Banerjee, S.K., Pogolotti, A., and Rupley, J.A. (1975) J. Biol. Chem. 250, 8260-8266). Our results indicate that the resonance of Cgamma or Trp-62 is a convenient monitor of lysozyme self-association. The chemical shift of this resonance reflects the extent of aggregation, while the line width yields information about the lifetime of the intermolecular contact. This lifetime is 1 to 2 ms at 38 degrees (9 mM protein, 0.1 M NaCl, pH 7). Our results also indicate that self-association of lysozyme is not accompanied by any general conformational change, and that binding of a lanthanide ion (at the metal ion binding site near the carboxylate groups of ASP-52 AND Glu-35) strongly suppresses self-association.     

Studies of chemically modified histidine residues of proteins by carbon 13 nuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodoacetate.

It is shown that natural abundance 13C NMR spectroscopy can be used to determine the structures and relative amounts of chemically modified forms of a histidine residue of a peptide or protein. The unfractionated product of the reaction of N alpha-acetyl-L-histidine with bromoacetate yields four resonances of nonprotonated aromatic carbons. These resonances are assigned (on a one-to-one basis) to C gamma of the intact amino acid, the two monocarboxymethylated derivatives (at N delta1 and N epsilon2), and the dicarboxymethylated derivative. The effect of pH on the chemical shift of C gamma is characteristic for each of the four species. This property is used to study the carboxymethylation of His-15 of hen egg white lysozyme upon treatment with iodoacetate. With the use of various reaction conditions, His 15 is carboxymethylated in detectable quantities only at N epsilon2. The spectra of the various reaction mixtures indicate which conditions are best for maximizing the yield of this derivative. A comparison of the spectrum of chromatographically pure [N epsilon2-carboxymethylhistidine-15]lysozyme with that of the intact protein indicates that the chemical modification does not significantly affect the conformation of the protein (at least in the regions of all aromatic amino acid residues).     

Nuclear-magnetic-resonance studies of eukaryotic cytochrome c. Assignment of resonances of aromatic amino acids

The aromatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double-resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X-ray structure of cytochrome c. 33 resonances arising from 39 aroumatic protons of ferrocytochrome c, and 18 resonances arising from 27 aromatic protons of ferricytochrome c have been assigned.     

Nuclear-magnetic-resonance studies of eukaryotic cytochrome c. Assignment of resonances of aliphatic amino acids

The aliphatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double-resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X-ray structure of cytochrome c. There are eight firmly assigned methyl resonances of ferrocytochrome c and seven firmly assigned methyl resonances of ferricytochrome c.     

Protein selectivity in immobilized metal affinity chromatography based on the surface accessibility of aspartic and glutamic acid residues

The interaction of different species variants of cytochrome c and myoglobin, as well as hen egg white lysozyme, with the hard Lewis metal ions Al³⁺, Ca²⁺, Fe³⁺, and Yb³⁺ and the borderline metal ion Cu²⁺, immobilized to iminodiacetic acid (IDA)-Sepharose CL-4B, has been investigated over the rangepH 5.5–8.0. With appropriately chosen buffer and metal ion conditions, these proteins can be bound to the immobilized M ⁿ +-IDA adsorbents via negatively charged amino acid residues accessible on the protein surface. For example, tuna heart cytochrome c, which lacks surface-accessible histidine residues, readily bound to the Fe³⁺-IDA adsorbent, while the other proteins also showed affinity toward immobilized Fe³⁺-IDA adsorbents when buffers containing 30 mM of imidazole were used. These studies document that protein selectivity can be achieved with hard-metalion immobilized metal ion affinity chromatography (IMAC) systems through the interaction of surfaceexposed aspartic and glutamic acid residues on the protein with the immobilized M ⁿ +-IDA complex. These investigations have also documented that the so-called soft or borderline immobilized metal ions such as the Cu²⁺-IDA adsorbent can also interact with surface-accessible aspartic and glutamic acid residues in a protein-dependent manner. A relationship is evident between the number and extent of clustering of the surfaceaccessible aspartic and glutamic acid residues and protein selectivity with these IMAC systems. The use of elution buffers which contain organic compound modifiers which replicate the carboxyl group moieties of these amino acids on the surface of proteins is also described.Abbreviations IDA iminodiacetic acid - IDA-Mⁿ⁺ iminodiacetic acid chelated to metal ion - IMAC immobilized metal affinity chromatography - DHCC dog heart cytochrome c - HHCC horse heart cytochrome c, THCC, tuna heart cytochrome c - HMYO horse skeletal muscle myoglobin - SMYO sheep skeletal muscle myoglobin - HEWL hen egg white lysozyme     

Re-evaluation of intramolecular long-range electron transfer between tyrosine and tryptophan in lysozymes. Evidence for the participation of other residues.

One-electron oxidation of six different c-type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gamma- and pulse-radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120 x s(-1) to 1000 x s(-1) at pH 7.0 and room temperature [extremes are hen and turkey egg white (120 x s(-1)) and human milk (1000 x s(-1))]. In hen and turkey egg white lysozymes we show that another reactive site is the Asn103-Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis.     

Solution structure of mitochondrial cytochrome c. II. 1H nuclear magnetic resonance of ferrocytochrome c

The 1H nuclear magnetic resonance spectrum of tuna ferrocytochrome c has been studied and the resonances of all 49 amino acid methyl groups have been assigned to specific absorption lines. In comparison with resonance assignments in the ferricytochrome c spectrum, the secondary shifts of resonances of ferrocytochrome c are smaller and the identification of characteristic spin-systems from comparison of spectra from homologous proteins more difficult. For this reason, two-dimensional nuclear magnetic resonance exchange correlated spectroscopy has been used to correlate the assigned resonances of tuna ferricytochrome c with previously unassigned resonances of tuna ferrocytochrome c.     

Nuclear-magnetic-resonance studies of ferrocytochrome c. pH and temperature dependence

The pH dependence and the temperature dependence of the nuclear magnetic resonance spectrum of horse ferrocytochrome c are described. This protein is very stable; it maintains an ordered structure over the pH range 4 to 12 at 25 degrees C and over the temperature range 4 degrees C to 97 degrees C at pH 7.0. The dynamic characteristics of the conformation of ferrocytochrome c were investigated. Particular emphasis was laid on the aromatic resonances and resonances of methyl groups shifted far upfield. Tyr-48 and Phe-46 were found to be relatively immobile whilst a region of the protein close to Ile-57 was found to be relatively flexible.     

13C-NMR studies of horse ferrocytochrome c. Assignment and temperature dependence of methyl resonances

The 13C and proton chemical shifts of 53 of the 55 methyl resonances of horse ferrocytochrome c have been determined by editing natural abundance 13C spectra according to the number of attached protons, observing the temperature dependence of the chemical shifts, and correlating 13C and proton chemical shifts in two-dimensional spectra. Previous assignments of proton shifts allow 16 of the 13C resonances to be assigned firmly.     

Complete assignment of carbon signals in a stereospecific peptide via selective and single off-resonance proton decoupling experiments. Analysis of the carbon-13 nuclear magnetic resonance spectrum of alumichrome at 67.88 MHz.

Polypeptides and proteins in native conformation exhibit 13C NMR spectra which are highly nondegenerate. Assignment of resonances to carbons in particular residues is hence a prerequisite for a structural analysis of the spectroscopic data. For nonprotonated carbonyl carbons, the assignment can be achieved by selective (1H alpha)13C' 2J decoupling. Using this method, we have assigned the Orn1 and Gly2 carbonyl resonances in alumichrome at 67.9 MHz. We show that a single off-resonance experiment with the decoupling frequency centered in the aliphatic proton spectrum is sufficient to assign unequivocally all the protonated carbon resonances via analysis of the reduced 1J heteronuclear splittings. Alumichrome thus becomes the first complex polypeptide spin system whose 1H, 15N, and now 13C nuclear resonances have been fully identified to date. 13C chemical shifts and 1H--13C spin--spin couplings are discussed in terms of structural strain leading to specific orbital hybridizations and on the basis of polarization effects due to electron density shifts toward hydrogen-bonding and metal-binding sites. A number of 3J(13C--C--C--1H) coupling constants measured on selected multiplets after resolution enhancement were used to derive the x-related Karplus relationship 3J(theta) = (10.2 cos2 theta -- 1.3 cos theta + 0.2) Hz.     

Studies of individual carbon sites of azurin from Pseudomonas aeruginosa by natural-abundance carbon-13 nuclear magnetic resonance spectroscopy.

The environments of the aromatic residues (and of the single arginine residue) of azurin from Pseudomonas aeruginosa are investigated by means of natural-abundance 13C Fourier transform NMR spectroscopy. In the case of the diamagnetic Cu(I) azurin, all 17 nonprotonated aromatic carbons (and Czota of Arg-79) yield narrow resonances. Furthermore, a single-carbon amide carbonyl resonance with an unusual chemical shift (peak chi) is observed. The pH dependence of chemical shifts is used to identify the resonances of Cgamma of titrating histidines, and of Cgamma and Czota of the two tyrosines. The resonances of Cgamma and Cdelta2 of the single tryptophan residue (and Czota of Arg-79) are also identified. The pKa values of the two tyrosines are different from each other and higher than typical values of "solvent-exposed" tyrosine residues. Two of the four histidine residues do not titrate (in the pH range 4 to 11). The resonance of Cgamma of one histidine exhibits a pH titration with fast proton exchange behavior and a pKa of 7.5 +/- 0.2. The direction of the titration shift indicates that the imidazole form of this histidine is the Ndelta1-H tautomer. The Cgamma resonance of the other titrating histidine exhibits slow exchange behavior with a pKa of about 7. The imidazole form of this histidine is the Nepsilon2-H tautomer. When going to the paramagnetic Cu(II) protein, only 11 of the 19 carbons mentioned above yield resonances that are narrow enough to be detected. Also, some of the observed resonances exhibit significant paramagnetic broadening. A comparison of spectra of fully reduced azurin, mixtures of reduced and oxidized azurin, and fully oxidized azurin yields the following information. (i) Peak chi arises from an amide group that probably is coordinated to the copper. (ii) The two nontitrating histidine residues are probably copper ligands, with Ndelta1 coordinated to the metal. (iii) The side chains of Arg-79 and the two tyrosine residues are not coordinated to the copper, and Trp-48 is probably not a ligand either. (iv) The gamma carbons of Trp-48, the tyrosine with the lower pKa, the titrating histidine with slow exchange behavior, and three or four of the six phenylalanine residues are sufficiently close to the copper to undergo significant paramagnetic broadening in the spectrum of oxidized azurin.     

Mini-myoglobin. The structural significance of haem-ligand interactions

The properties of purified mini-myoglobin, the fragment 32-139 of horse heart myoglobin reconstituted with protohaem, have been investigated from a structural and functional view point. The recovery of secondary structure observed in the carbon monoxide derivative of mini-myoglobin, as shown by circular dichroism, and the overall similarity of the haem pocket to that of myoglobin, as deduced from the fluorescence properties of the complex with 1-anilino-8-naphthalene sulphonate, indicate that, in the presence of the constraints imposed by the haem and its ligands, the miniprotein reacquires a conformation close to that of native myoglobin. These spectroscopic data parallel the conclusions drawn from the results of ligand combination and dissociation kinetics; stopped-flow experiments indicate that carbon monoxide and oxygen bind to mini-myoglobin with rates almost identical with those of myoglobin itself. The significance of mini-myoglobin as a model of an oxygen-carrying protein, with some of the expected functional characteristics of an ancestor haemoprotein, is discussed, with reference to the mosaic structure of the myoglobin gene and the role of different exons in the evolution of proteins.     

Heterologous Expression of Hen Egg White Lysozyme and Resonance Assignment of Tryptophan Side Chains in its Non-native States

A new protocol is described for the isotope (¹⁵N and ¹³C,¹⁵N) enrichment of hen egg white lysozyme. Hen egg white lysozyme and an all-Ala-mutant of this protein have been expressed in E. coli. They formed inclusion bodies from which mg quantities of the proteins were purified and prepared for NMR spectroscopic investigations. ¹H,¹³C and ¹⁵N main chain resonances of disulfide reduced and S-methylated lysozyme were assigned and its residual structure in water pH 2 was characterized by chemical shift perturbation analysis. A new NMR experiment has been developed to assign tryptophan side chain indole resonances by correlation of side chain and backbone NH resonances with the C^γ resonances of these residues. Assignment of tryptophan side chains enables further residue specific investigations on structural and dynamical properties, which are of significant interest for the understanding of non-natives states of lysozyme stabilized by hydrophobic interactions between clusters of tryptophan residues.     

Proton magnetic resonance spectroscopic studies of proteins containing deuterated tryptophan residues

Summary The deuteration of the tryptophan residues of hen egg white lysozyme, bovine-lactalbumin and bovine-lactoglobulin in d-TFA has been studied by PMR spectroscopy. It is found that short times of exposure to d-TFA allow selective deuteration at the C-2 position with only a small amount of deuteration at the C-5 position, as expected from studies on model peptides described in the previous paper. The proteins studied essentially regained their native structures after the treatment, except for broadening and shifting of the histidine resonances in the case of-lactalbumin. Selective deuteration at the tryptophan C-2 position was readily observed by difference spectroscopy of the denatured protein, but PMR difference spectra of the same proteins in benign solvents did not contain resonances from all of the exchanged protons. Some resonances could not be observed because of line broadening, which causes the resonances to fall below the limit of sensitivity of detection at 100 MHz. Deuteration by brief exposure to d-TFA should be useful for the identification of tryptophan resonances in the PMR spectra of native proteins.The deuteration of all the aromatic protons of tryptophan residues in proteins by immersion in d-TFA for 4 hours at room temperature was studied. This technique is unlikely to be of general use for the simplification of the aromatic region of the PMR spectra of native proteins because of the degradation of tryptophan residues which results from the acid treatment.An invited article.     

1H-NMR studies of structural homologies between the heme environments in horse cytochrome c and in cytochrome c-552 from Euglena gracilis

With the use of proton-proton Overhauser enhancement experiment the spatial arrangement relative to the heme group of amino acid side chains in the heme crevice of horse ferrocytochrome c and ferrocytochrome c-552 from euglena gracilis was investigated. From these data and the known crystal structure for mammalian cytochromes c, individual assignments were obtained for several aromatic residues in horse ferrocytochrome c. This then provided a basis for delineating homologies between the polypeptide conformations near the heme group in horse ferrocytochrome c and ferrocytochrome c-552, for which no crystal structure has as yet been described.     

Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo.

The titration behavior of individual tyrosine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of Czeta and Cgamma of these residues in natural abundance of 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes, at 37 degrees) of cyanoferrimyoglobins from sperm whale, horse, and red kangaroo. A comparison of the pH dependence of the spectra of the three proteins yielded specific assignments for the resonance of Tyr-151 (sperm whale) and Tyr-103 (sperm whale and horse). Selective proton decoupling yielded specific assignments for Czeta of Tyr-146 of the cyanoferrimyoglobins from horse and kangaroo, but not the corresponding assignment for sperm whale. The pH dependence of the chemical shifts indicated that only Tyr-151 and Tyr-103 are titratable tyrosine residues. Even at pH 12, Tyr-146 did not begin to titrate. The titration behavior of C zeta and Cgamma of Tyr-151 of sperm whale cyanoferrimyoglobin yielded a single pK value of 10.6. The pH dependence of the chemical shift of each of the resonances of Tyr-103 of the cyanoferrimyoglobins from horse and sperm whale could not be fitted with the use of a single pK value, but was consistent with two pK values (about 9.8 and 11.6). Furthermore, the resonances of Czeta and Cgamma of Tyr-103 broadened at high pH. The titration behavior of the tyrosines of sperm whale carbon monoxide myoglobin and horse ferrimyoglobin was also examined. A comparison of all the experimental results indicated that Tyr-151 is exposed to solvent, Tyr-146 is not exposed, and Tyr-103 exhibits intermediate behavior. These results for myoglobins in solution are consistent with expectations based on the crystal structure.     

Spectral evidence for sub-picosecond iron displacement after ligand detachment from hemoproteins by femtosecond light pulses.

We have measured spectral and kinetic differences in protoheme, sperm whale or horse heart myoglobin and human hemoglobin following photodissociation induced by optical pulses of 80 fs duration. Full ligation was performed with oxygen or carbon monoxide. Femtosecond kinetics and transient difference spectra revealed the appearance of a deoxy species with tau approximately equal to 250-300 fs. The transient deoxy species in myoglobin and hemoglobin evidenced a 3-4 nm red shift of their delta A spectra compared with the equilibrium delta A spectrum. This shift was not observed after photodissociation of the carbon monoxide liganded protoheme. We proposed that the 250 fs time constant corresponding to the appearance of the deoxy-like species is related to the displacement of the ferrous iron out of the heme plane. Consequently, the small red shift of the delta A spectra observed in photodissociated hemoproteins may be tentatively attributed to changes in the vibrational modes of either the proximal histidine-Fe2+ bond and/or of the N4 porph-Fe-N epsilon His (F8) bent.